Lyme disease is the most common tick-borne illness in the United States and Europe. It is caused by Borrelia burgdorferi, a bacterial pathogen that is maintained in nature in a zoonotic cycle between various species of small mammals and an ixodid tick vector. A hallmark of the Lyme disease spirochete is its unusual segmented genome, which includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded functions are critical for successful adaptation to the different environments that B. burgdorferi encounters during its infectious cycle. We have developed genetic tools to investigate basic aspects of the unusual genomic organization, cellular structure and metabolism of B. burgdorferi. We have extended this investigation to an in vivo setting with an experimental system that closely mimics the natural arthropod vector/rodent host infectious cycle. Through an understanding of the basic molecular biology of the organism, we hope to gain insight into the infectious strategy utilized by this significant vector-borne pathogen and thereby facilitate efforts to prevent, diagnose and treat Lyme disease. In FY2019 we described the novel application to spirochetes of the FlAsH technique for fluorescently labeling specific proteins in living cells (1). This technique entails engineering a tetracysteine motif in the coding sequence of a protein of interest and staining with a membrane-permeable biarsenical dye that binds to this motif. The small increase in protein size conferred by the 6 amino acid motif minimizes potential negative effects that can occur when targets are fused to larger fluorogenic proteins, such as steric hindrance, improper protein folding, incorrect cellular location, or inappropriate oligomerization. This technique worked well for labeling both inner and outer spirochetal membrane proteins, indicating that the dye is freely diffusible across the intact outer membrane of living spirochetes without the need for fixation or permeabilization. Labeled spirochetes were quantitatively detected by fluorescence microscopy and flow cytometry. We found that concatenating two copies of the tetracysteine motif increased the intensity and duration of fluorescence. Genes encoding tetracysteine-tagged proteins were expressed from endogenous loci or in trans from shuttle vectors, indicating that single or multicopy expression sites are effective options. We successfully applied this technique to the distantly related spirochetes B. burgdorferi, a zoonotic human pathogen, and L. biflexa, a free-living saprophyte, suggesting that the FlAsH system can be applied broadly across all spirochete species. Mouse-tick infection studies of tetracysteine-tagged B. burgdorferi demonstrated that the tetracysteine motif was stably maintained in vivo and did not adversely affect infectivity in either the arthropod vector or murine host. Finally, biarsenical-bound proteins could be followed over several days, indicating that the FlAsH dye approach can be used in time-course studies of live cells (1). Genetic manipulation of B. burgdorferi is currently extremely inefficient, requiring microgram quantities of DNA, yet yielding only a few transformants. This severely limits the application of effective genetic screens to the Lyme disease spirochete. Endogenous plasmid-encoded restriction/modification (R/M) systems constitute part of the barrier to stable introduction of foreign DNA in B. burgdorferi. In FY2019, we continued a long-standing collaboration with Dr. Gang Fang at Mt. Sinai School of Medicine, NY. We have identified the DNA sequence motifs recognized by R/M systems of the widely used B. burgdorferi type strain B31 and extended this analysis to prototypic B. garinii and B. afzelii strains, which are agents of Lyme borreliosis in Europe. Armed with this information, Dr. Jenny Wachter, a postdoctoral fellow in MGS, has designed shuttle vectors and selectable markers that lack these R/M sites. Additionally, in collaboration with Dr. Craig Martens and Stacy Ricklefs of the RTB at RML, Jenny has completed RNA-seq analysis of strains containing or lacking R/M genes and found limited evidence for epigenetic regulation of gene expression in B. burgdorferi. Ongoing studies will assess the utility of sequence-optimized constructs for efficient transformation of B. burgdorferi and explore their potential to expand genetic studies in the Lyme disease spirochete. In FY2019 we assisted Christina Savage, William Arnold and Brandon Jutras, current and former graduate students in Dr. Brian Stevenson's lab at the University of Kentucky, with an investigation of the role of the BpuR DNA- and RNA-binding protein of B. burgdorferi in the modulation of spirochete physiology (2). Our contribution to this study comprised an analysis of bpuR transcript levels in the spirochete during acquisition, persistence and transmission of B. burgdorferi by the tick vector. In FY2019, Dr. Rosa completed a sabbatical in the laboratory of Dr. Christine Jacobs-Wagner at Yale University. During FY2019, Dr. Rosa contributed to a Jacobs-Wagner lab study that resulted in the development of a number of new molecular tools for B. burgdorferi, including several new fluorescent proteins, promotors of varying strength for modulated levels of gene expression, and novel selectable markers. (3) Dr. Rosa also assisted members of the Jacobs-Wagner lab in an investigation of how Borrelia replicates and segregates its highly segmented genome, as required for the maintenance and survival of B. burgdorferi in nature and ultimately central to transmission of Lyme disease. Finally, Dr. Rosa took advantage of the expertise of the Jacobs-Wagner lab to fluorescently tag and computationally analyze the cellular locations of a recently described set of bacterial cytoskeletal elements termed bactofilins. This work was done in conjunction with an ongoing genetic analysis of the role of bactofilins in B. burgdorferi, conducted by Valentina Carracoi in Dr. Rosas lab at RML. Various aspects of the genome localization and bactofilin projects continue as a collaborative effort between the Jacobs-Wagner and Rosa. 1. Hillman, C., Stewart, P.E., Strnad, M., Stone, H., Starr, T., Carmody, A., Evans, T.J., Carracoi, V., Wachter, J., and Rosa, P.A., Visualization of spirochetes by labeling membrane proteins with fluorescent biarsenical dyes. Front. Cell. Infect. Microbiol., in press 2019. 2. Jutras, B., Savage, C., Arnold, W., Lethbridge, K. Carroll, D., Tilly, K., Bestor, A., Zhu, H., Seshu, J., Zueckert, W., Stewart, P., Rosa, P., Brissette, C., and Stevenson, B. ,The Lyme disease spirochete's BpuR DNA- / RNA-binding protein is differentially expressed during the mammal-tick infectious cycle and affects translation of the SodA superoxide dismutase. Mol.Microbiol., in press 2019. 3. Takacs, C.N., Kloos, Z.A., Scott, M., Rosa, P.A., and Jacobs-Wagner, C., Fluorescent proteins, promoters and selectable markers for applications in the Lyme disease spirochete Borrelia burgdorferi. Appl. Environ. Microbiol. 84:e01824-18, 2018